Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

ABSTRACT: Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sit

SUBMITTER: Farquhar ER 

PROVIDER: S-EPMC10510565 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature